The presence of bacterial pathogens is a well recognized cause of various medical and etiological problems, so that there is an ever present need for the detection of bacterial pathogens in both clinical specimens (i.e. blood, tissue, urine and other body extracts and fluids) and agricultural specimens (such as food products).
However, current tests for the detection of bacterial pathogens, such as in food, typically require a number of days to complete. During this period of time, between sampling and assay determination, fresh food and dairy products will enter the food chain and therefore be consumed by the public. If a test indicates the presence of pathogens, expensive product recalls may result, or, worse, before the test results are discovered an outbreak of sickness may occur.
This was exactly the case in a number of recent outbreaks of Listeria in fresh dairy and vegetable products. In the case of Listeria, an outbreak generally produces a death rate greater than 40%. In addition this rate is much higher for newborns, pregnant women, the elderly and immunocompromised individuals. Spontaneous abortions result even in otherwise asymptomatic patients. In fact, it is estimated that as many as 2% of all spontaneous abortions may be due to Listeria infection.
As stated above, traditional methods to detect the presence of bacterial food pathogens require an extended period of time, basically due to the need for an incubation period. This incubation period is intended to allow for recovery of injured bacteria, growth of these bacteria from a background of competing microorganisms and an increase in bacterial cell numbers to more readily aid in identification. In many cases a series of two or three separate incubations is needed to isolate the target bacteria.
In the standard FDA procedure for detection of Listeria in food products (Bacteriological Analytical Manual, 6th ed., 1984; Supplement, September 1987, Chapter 29) 25 g or 25 ml of a food sample is mixed with 225 ml of enrichment broth. This sample in broth mixture is incubated for 7 days. At the end of days 1 and 7 a sample of the broth culture is streaked onto petri plates containing selective growth agar and these plates are incubated for an additional 2 days. Identification of Listeria colonies confirms the presence of Listeria in the original food sample. This identification, however, is subjective, and therefore prone to misinterpretation, and this procedure requires a minimum of 7 days to confirm Listeria negative samples.
These conventional methods are slow, requiring 5-14 days, or more, depending upon the bacteria species of interest. In a given food sample it is also possible to miss target bacteria (false negatives) due to many reasons including inadequate incubation conditions (time, growth media), subjectivity of the person interpreting the results, identification on petri plates, over-growth by competing organisms or a combination of these and other problems.
More recent methods of bacterial detection in food products have utilized immunoassays. Antibodies to an antigen present in the bacteria of interest are generally used in these methods in some form of a two site assay. That is, one antibody is immobilized and acts to capture the target bacterial antigens. This allows for separation of the target antigen from the food sample. A second antibody to this antigen (having the same or a different epitope) is labeled in some fashion such as radioactively with I.sup.125 or enzymatically with horse radish peroxidase, and when added to the immobilized antibody antigen complex is also immobilized. Subsequent steps remove unbound labeled antibody. The label left attached is measured and usually compared against standards (positive and negative controls) to determine the presence of the target bacteria. At least one of the two antibodies used in the two site assay must be specific to the target bacteria. This type of immunoassay is known as a direct assay. Other forms, such as the competition assay are also used but tend to be less sensitive.
Because of the actual sensitivity limit of these assays it remains necessary to culture the target bacteria from the food sample. In some of the newer immunoassay tests incubation times have been reduced and the number of separate incubation steps have also been reduced. The resulting tests, however, still require about 48 hours to complete. Many of the problems associated with culturing the target organisms, as described above, also remain.
In a generalized "rapid" immunoassay for detection of Listeria, 25 g or 25 ml of food sample is mixed with 225 ml of enrichment broth, as in the FDA procedure. This culture mixture is incubated for 24 hours. From this 250 ml of culture, 1 ml is removed and added to 9 ml of selective growth medium. This selective culture mixture is incubated for an additional approximately 24 hours. At this point, depending upon the actual immunoassay format, some fraction of the total 10 ml subculture (usually 0.2 ml-1.0 ml) is tested by immunoassay for the presence of Listeria.
In summary, these existing "rapid" immunoassay procedures for bacterial detection in food samples all require at least one (usually two or more) dilution of sample step (into growth medium) followed by an assay procedure which only utilizes a fraction of this final culture. The actual assay sample thus only corresponds to a small fraction of the original food sample. The bacterial culture step, or steps, must therefore overcome this dilution factor, adding to the amount of needed culture time.
These and other disadvantages of the prior art methods are overcome by the present invention which provides a fast and accurate method for the detection of Listeria.